Anti-mast cell antibody

Abstract

The invention relates to a method for depleting mast cells. The invention also relates to a method of treating mast cell disease in a subject in need thereof. The method involves administering an anti-mast cell antibody wherein the antibody is administered by a slow absorption delivery pathway, in particular wherein the antibody is administered subcutaneously. Administration of anti-mast cell antibodies via a slow absorption delivery pathway (e.g., subcutaneous or intradermal) can avoid allergic reactions occurring when anti-mast cell antibodies are administered via other pathways (e.g., intravenous).

Description

Technical Field

[0001] This invention relates to methods for depleting mast cells. It also relates to methods for treating mast cell diseases in subjects with this need. The method involves administering an anti-mast cell antibody, wherein the antibody is administered via a slow absorption delivery route, particularly wherein the antibody is administered subcutaneously. Background Technology

[0002] Mast cells are inflammatory immune sentinel cells found throughout the body's tissues, particularly at interfaces with the external environment (e.g., on the mucosal surfaces of the intestines and lungs), in the skin, and around blood vessels. Mast cells play crucial roles in many physiological functions, such as vasodilation, wound healing, and angiogenesis. Most importantly, they play a central role in the body's adaptive immune response. Given their importance in inducing inflammatory cascades, mast cells, when activated, also participate in driving many allergic, inflammatory, and pruritic conditions. Mast cells can be activated by a variety of endogenous and exogenous stimuli, including allergen-specific IgE autoantibodies, complement, toll-like receptor (TLR) agonists, alarmin, cytokines, neuropeptides, drugs, and venom. Following stimulation, mast cells release pre-formed granule storage mediators (e.g., histamine, trypsin, chymotrypsin, and TNF-α) into the extracellular space—a process known as degranulation. This is followed by a second wave of de novo synthesis of prostaglandins and leukotrienes, ultimately leading to a rapid inflammatory response. The best-investigated mechanism for mast cell activation is through IgE-mediated FcεRI crosslinking, which is highly expressed on human mast cells. (Except...) In addition, human mast cells also express IgG Fc receptors (FcγR), complement protein receptors, toll-like receptors (TLR), c-KIT receptors, cytokine receptors, and G protein-coupled receptor MRGPRX2. All of these receptors are involved in mast cell activation and immune responses.

[0003] Mast cell disorders are characterized by undesirable mast cell degranulation. This may be due to an increased sensitivity of the patient's mast cells to activation by external stimuli. The most common is type I hypersensitivity, such as urticaria (hives), which occurs due to an excessive IgE-mediated immune response to antigens that should not normally trigger the immune system. Another mast cell disorder is mastocytosis, which is characterized by an excessive number of mast cells in the body. Mastocytosis is often associated with mutations in the c-kit gene encoding the c-KIT receptor, particularly gain-of-function mutations that lead to constitutive c-KIT activation. The abnormally high number of mast cells found in patients with mastocytosis and their potential to release large amounts of histamine into the bloodstream means that patients with this disease are at a much higher risk of developing severe allergic reactions, such as anaphylactic reactions.

[0004] Numerous treatments for mast cell diseases have been proposed. Therapies (such as anti-IgE antibodies like omalizumab) aim to inhibit specific mast cell triggers. Other therapies target mast cell mediators; for example, antihistamines are first-line treatments for many mast cell diseases. However, there is evidence that a more comprehensive inhibition of mast cell number or activity, rather than targeting a single trigger or mediator, yields broader efficacy. Since c-KIT signaling is involved in mast cell differentiation, maturation, and survival, tyrosine kinase inhibitors (TKIs) that inhibit the c-KIT receptor have also been developed. However, some drawbacks of c-KIT-targeting TKIs include undesirable reactivity to other related kinases and the emergence of TKI-resistant mast cells.

[0005] Recently, anti-c-KIT antibodies have been developed to achieve more selective targeting of mast cells. However, mast cells have proven to be a particularly difficult cell type to target with therapeutic antibodies due to their high expression of Fc receptors. This means they are highly susceptible to participating in adaptive immune responses induced by the antibody itself. Therefore, a significant concern with systemic administration of c-KIT-targeting antibodies is the potential for mast cell activation and degranulation induced by the antibody's Fc domain via c-KIT-dependent aggregation of mast cell FcγR. Consequently, barzolvolimab, an anti-c-KIT antibody targeting Fc-dead cells, has been engineered and is currently in a phase 2 trial for the treatment of chronic spontaneous urticaria (Alvarado et al. 2022; Basic and Translational Allergy Immunology; 77:2393-2403). Bazolimumab binds to c-KIT and blocks SCF-dependent signaling, but importantly, the antibody does not participate in the innate immune system because it has been engineered to eliminate this function using a key mutation in the antibody's Fc portion, thereby preventing the involvement of FcγR. Summary of the Invention

[0006] As reported in this paper, "Fc-deficient" anti-mast cell antibodies have a very limited ability to deplete mast cells. This is due to the lack of Fc effector function, such as antibody-dependent cytotoxicity mediated by the binding of the antibody's Fc region to Fc receptors on immune cells, including mast cells themselves.

[0007] Therefore, the inventors sought to reintroduce Fc effector function into anti-mast cell antibodies to enhance mast cell depletion while avoiding undesirable mast cell activation and anaphylactic reactions caused by Fc receptor binding. As demonstrated herein, effective mast cell depletion and reduced anaphylactic reactions can be achieved by optimizing the administration route of anti-mast cell antibodies. More specifically, administration of anti-mast cell antibodies via slow absorption delivery routes (e.g., subcutaneous or intradermal) avoids the anaphylactic reactions that occur with administration via other routes (e.g., intravenous).

[0008] By employing a slow absorption delivery pathway, it is believed that the slow absorption of anti-mast cell antibodies into the patient's body fluids (lymph or blood) will lead to a relatively slow depletion of mast cells. Not wanting to be bound by theory, it is thought that the slow depletion rate results in a slower release of histamine and other chemical mediators after mast cell lysis. This, in turn, reduces the risk of severe allergic reactions (such as anaphylactic reactions) in patients with mast cell disease.

[0009] In a first aspect, the present invention provides a method for depleting mast cells in a subject, the method comprising administering an effective amount of an anti-mast cell antibody to the subject, wherein the anti-mast cell antibody is administered to the subject via a slow absorption delivery route.

[0010] In a second aspect, the present invention provides a method for treating mast cell disease in a subject in need, the method comprising administering to the subject a therapeutically effective amount of an anti-mast cell antibody, wherein the anti-mast cell antibody is administered to the subject via a slow absorption delivery route.

[0011] In some implementations, the anti-mast cell antibody is administered subcutaneously.

[0012] In some implementations, anti-mast cell antibodies are administered via a subcutaneous implant.

[0013] In some implementations, the anti-mast cell antibody is administered intradermally.

[0014] In some implementations, the anti-mast cell antibody is not administered intravenously.

[0015] In some embodiments, the anti-mast cell antibody specifically binds to proteins expressed on the surface of mast cells. In some embodiments, the anti-mast cell antibody is an anti-c-KIT antibody. In some preferred embodiments, the anti-mast cell antibody comprises an Fc region. In some embodiments, the Fc region has one or more Fc effector functions. In some embodiments, the Fc region has one or more enhanced Fc effector functions relative to the wild-type Fc region. In some embodiments, the one or more Fc effector functions are selected from: antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC). In some embodiments, the Fc region has the Fc effector function antibody-dependent cytotoxicity (ADCC). In some embodiments, the Fc region has enhanced Fc effector function antibody-dependent cytotoxicity (ADCC) relative to the wild-type Fc region.

[0016] In some embodiments, the anti-mast cell antibody is a human IgG antibody. In some embodiments, the Fc region contains the amino acid substitution S239D / I332E. In some embodiments, the Fc region is non-fucosylated. In some embodiments, the Fc region is galactosylated.

[0017] In some implementations, mast cell disease is mastocytosis. In some implementations, the subject has an abnormally high number of mast cells. In some implementations, the subject is a human being.

[0018] In some embodiments, the anti-mast cell antibody is formulated in a pharmaceutical composition. In some embodiments, the pharmaceutical composition is formulated for the slow absorption of the anti-mast cell antibody, optionally wherein the pharmaceutical composition is a modulated release formulation selected from slow-release, extended-release, prolonged-release, sustained-release, delayed-release, or controlled-release.

[0019] In some embodiments, the method reduces or prevents mast cell activation or mast cell degranulation. In some embodiments, the method reduces or prevents allergic or anaphylactic reactions. In some embodiments, administration of anti-mast cell antibodies does not induce mast cell degranulation, allergic reactions, and / or anaphylactic reactions.

[0020] In a third aspect, the present invention provides an anti-mast cell antibody for a method of treating mast cell disease, wherein the method comprises administering a therapeutically effective amount of the anti-mast cell antibody to a subject, wherein the anti-mast cell antibody is administered to the subject via a slow absorption delivery route.

[0021] In a fourth aspect, the present invention provides the use of anti-mast cell antibodies in the preparation of medicaments for treating mast cell diseases, wherein the treatment comprises administering a therapeutically effective amount of the anti-mast cell antibody to a subject, wherein the anti-mast cell antibody is administered to the subject via a slow absorption delivery route. Attached Figure Description

[0022] Figure 1 The results of the desensitization experiment observed in wild-type mice administered with intraperitoneal injection of anti-c-KIT antibody are shown. (A) Mice were administered escalating doses (0.1, 0.3, 1.0, 3.0, and 10 μg / injection) of anti-c-KIT antibody with enhanced ADCC activity (ADCC + anti-CD117), ADCC + isotype control, or PBS control every 30 minutes, and the degree of anaphylactic reaction (i.e., decrease in body temperature) was measured. (B) Following the desensitization phase, the same mice were challenged again with larger doses of anti-c-KIT antibody on days 0, 2, and 4, and body temperature was measured. (C) On day 7, mast cells in the peritoneal cavity were quantified.

[0023] Figure 2The degree of anaphylactic response and the release of mast cell proteases (MCPT-6 and MCPT-1) are shown in mice that received 30 μg of anti-c-KITADCC+ antibody or anti-c-KIT Fc loss-of-function antibody via intraperitoneal (B), intravenous (C), or subcutaneous (D) injection. One hour after antibody challenge, mice were euthanized, blood samples were collected, and serum was separated to quantify the release of mast cell-specific proteases, as described in (A).

[0024] Figure 3 The study showed that intraperitoneal injection of anti-c-KIT ADCC+ antibody or anti-c-KIT wild-type antibody depleted mast cells in the peritoneal cavity (B), but induced an allergic reaction (C). As described in (A), mast cell count was evaluated on day 7 following intraperitoneal administration of the antibody.

[0025] Figure 4 The effects of three doses of anti-c-KIT antibody (Fc loss-of-function, WT, and ADCC+) administered subcutaneously on days 0, 2, and 4 on allergic reactions and mast cell depletion are shown in (A). As shown in (B), subcutaneous administration of anti-c-KIT antibody did not induce an allergic reaction. On day 7 post-administration, the number of mast cells in the peritoneal cavity (C), back skin (D), ear skin (E), and mesentery (F) was quantified.

[0026] Figure 5 The effects of different doses of anti-c-KIT ADCC+ antibody on mast cell number are shown. (A) Mice were administered subcutaneous doses of 1, 5, 10, or 30 μg of antibody on day 0 and day 7, and were then euthanized on day 14. (B) The number of mast cells in the peritoneal cavity was quantified.

[0027] Figure 6 The efficacy of anti-c-KIT antibodies (Fc loss-of-function, WT, and ADCC+) on mast cell numbers in different organs is shown. (A) Mice were given a subcutaneous dose of 30 μg of antibody on day 0 and again on day 7, and then euthanized on day 14. (B) and (C) show the mast cell profile in the peritoneal cavity. (D) shows the number of mast cells in the mesentery, dorsal skin, ear skin, lung, small intestine, and large intestine.

[0028] Figure 7 The effects of anti-c-KIT antibodies (Fc loss-of-function, WT, and ADCC+) on different leukocyte populations were demonstrated.

[0029] Figure 8 The effects of anti-c-KIT antibodies (Fc loss-of-function, WT, and ADCC+) on bone marrow progenitor cell populations were demonstrated.

[0030] Figure 9 This study demonstrates the role of antibody-induced mast cell exhaustion in reducing the severity of anaphylactic reactions in an IgE passive systemic anaphylactic model. Mice received three subcutaneous injections of anti-c-KIT antibodies (Fc loss-of-function, WT, or ADCC+) on days 0, 2, and 4, followed by IgE sensitization on day 7 and antigen challenge on day 8. Core body temperature was monitored one hour post-challenge to examine for hypothermia.

[0031] Figure 10 This study demonstrates the role of antibody-induced mast cell exhaustion in reducing the severity of anaphylactic reactions in an IgE passive systemic anaphylactic model. (A) Mice received two subcutaneous injections of anti-c-KIT antibodies (Fc loss-of-function, WT, or ADCC+) on days 0 and 7, followed by IgE sensitization on day 14 and antigen challenge on day 15. (B) Core body temperature was monitored one hour post-challenge to examine for hypothermia. (C) Blood was collected 60 minutes post-challenge for serum analysis of mast cell-specific proteases MCPT-1 and MCPT-6, followed by quantification of MCPT-1 and MCPT-6 by ELISA.

[0032] Figure 11 The effect of anti-c-KIT antibody (Fc loss-of-function and ADCC+) treatment on mrgprb2-mediated anaphylactic response is illustrated. (A) Mice received two subcutaneous injections of anti-c-KIT antibody on days 0 and 7, followed by a ciprofloxacin challenge on day 14. (B) Core body temperature was monitored one hour post-challenge to examine for hypothermia. (C) Blood was collected 60 minutes post-challenge for serum analysis of mast cell-specific proteases MCPT-1 and MCPT-6, followed by quantification of MCPT-1 and MCPT-6 by ELISA. Detailed Implementation

[0033] A. definition

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Without limiting any terminology, further clarification of some terms used herein is provided below.

[0035] As used herein, the term "immunoglobulin" includes polypeptides having a combination of two heavy chains and two light chains, regardless of whether they possess any associated specific immunoreactivity. "Antibody" refers to such a combination that has significant, known specific immunoreactivity against a target antigen. Both antibodies and immunoglobulins contain light and heavy chains, with or without interchain covalent bonds. The basic immunoglobulin structures in vertebrate systems are relatively well understood.

[0036] The general term "immunoglobulin" encompasses five distinct classes of antibodies that can be biochemically distinguished. Regarding IgG, immunoglobulins consist of two identical light polypeptide chains with a molecular weight of approximately 23,000 Daltons and two identical heavy chains with a molecular weight of 53,000 to 70,000 Daltons. These four chains are linked by disulfide bonds in a "Y" configuration, where the light chain begins at the mouth of the "Y" and continues through a variable region to bracket the heavy chain. The light chains of antibodies are classified as kappa or lambda (κ, λ). Each heavy chain class can bind to either a kappa or lambda light chain. Typically, the light and heavy chains are covalently bonded to each other, and when immunoglobulins are produced by hybridomas, B cells, or genetically modified host cells, the "tail" portions of the two heavy chains are linked together by covalent disulfide bonds or non-covalent bonds. In the heavy chain, the amino acid sequence extends from the N-terminus at the end of the Y-configuration fork to the C-terminus at the bottom of each chain. Those skilled in the art will understand that heavy chains are classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), among which there are several subclasses (e.g., γ1 to γ4). The properties of this chain determine the "class" of the antibody, namely IgG, IgM, IgA, IgD, or IgE. Immunoglobulin subclasses (isotypes) such as IgG1, IgG2, IgG3, IgG4, IgA1, etc., have been well characterized and are known to confer functional specialization. In view of this disclosure, those skilled in the art can readily identify modified forms of each of these classes and isotypes, and therefore they are within the scope of this invention.

[0037] The variable region of an antibody allows it to selectively recognize and specifically bind to epitopes on antigens. Specifically, the variable light (VL) and variable heavy (VH) domains of the antibody combine to form a variable region that defines a three-dimensional antigen-binding site. This quaternary antibody structure forms antigen-binding sites located at the ends of each arm of the Y-chain. More specifically, the antigen-binding site is defined by three complementary determining regions (CDRs) on each VH and VL chain.

[0038] The terms “variable region” and “variable domain” are used interchangeably herein and are intended to have equivalent meanings. The term “variable” refers to the fact that certain portions of the variable domains VH and VL in an antibody are widely different in sequence, and these portions are used for the binding and specificity of each particular antibody to its target antigen. However, variability is not uniformly distributed throughout the entire variable domain of the antibody. It is concentrated in three segments called “hypervariant loops” in each of the VL and VH domains, which form part of the antigen-binding site. The first, second, and third hypervariable rings of the Vλ light chain domain are referred to herein as L1(λ), ​​L2(λ), and L3(λ), and can be defined as containing residues 24 to 33 (L1(λ, consisting of 9, 10, or 11 amino acid residues), residues 49 to 53 (L2(λ, consisting of 3 residues), and residues 90 to 96 (L3(λ, consisting of 5 residues)) in the VL domain (Morea et al., Methods 20:267-279(2000)). The first, second, and third hypervariable rings of the Vκ light chain domain are referred to herein as L1(κ), L2(κ), and L3(κ), and can be defined as containing residues 25 to 33 (L1(κ, consisting of 6, 7, 8, 11, 12, or 13 residues), residues 49 to 53 (L2(κ, consisting of 3 residues), and residues 90 to 97 (L3(κ, consisting of 6 residues)) in the VL domain (Morea et al., Methods 20:267-279 (2000)). The first, second, and third hypervariable rings of the VH domain are referred to as H1, H2, and H3 in this paper, and can be defined as containing residues 25 to 33 (H1, consisting of 7, 8, or 9 residues), residues 52 to 56 (H2, consisting of 3 or 4 residues), and residues 91 to 105 (H3, highly variable in length) in the VH domain (Morea et al., Methods 20:267-279 (2000)).

[0039] Unless otherwise stated, the terms L1, L2, and L3 refer to the first, second, and third hypervariable rings of the VL domain, respectively, and cover hypervariable rings obtained from both Vκ and Vλ isoforms. The terms H1, H2, and H3 refer to the first, second, and third hypervariable rings of the VH domain, respectively, and cover hypervariable rings obtained from any known heavy chain isoform (including γ, ε, δ, α, or μ).

[0040] Hypervariable rings L1, L2, L3, H1, H2, and H3 may each contain a portion of a "complementary determinant region" or "CDR," as defined below. The terms "hypervariable ring" and "complementary determinant region" are not strictly synonymous, because the hypervariable ring (HV) is defined based on structure, while the complementary determinant region (CDR) is defined based on sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD., 1983), and the constraints of HV and CDR may differ in some VH and VL domains.

[0041] The CDRs of the VL and VH domains are generally defined as containing the following amino acids: residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in the light chain variable domain, and residues 31-35 or 31-35b (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in the heavy chain variable domain (Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Therefore, HV can be included within the corresponding CDR, and unless otherwise stated, the term "hypervariant ring" in the VH and VL domains should be interpreted as also encompassing the corresponding CDR, and vice versa.

[0042] The more conserved portion of the variable domain is called the framework region (FR), as defined below. The variable domains of the natural heavy and light chains each contain four FRs (FR1, FR2, FR3, and FR4, respectively), which predominantly adopt a β-sheet configuration and are linked by three hypervariable loops. The hypervariable loops in each chain are tightly held together by the FRs and, together with hypervariable loops from the other chain, contribute to the formation of the antibody's antigen-binding site. Structural analysis of the antibody has revealed the relationship between the sequence and shape of the binding site formed by the complementarity-determining region (Chothia et al., J. Mol. Biol. 227: 799-817 (1992)); Tramontano et al., J. Mol. Biol, 215:175-182 (1990)). Despite its high sequence variability, five of the six loops adopt only a small portion of the main chain conformation, termed the "typical structure." These conformations are primarily determined by the length of the ring, and secondarily by the presence of key residues at certain locations within the ring and frame regions, which determine the conformation through their ability to stack, hydrogen bond, or present unusual main-chain conformations.

[0043] As used herein, the term “CDR” or “complementarity-determining region” refers to a discontinuous antigen-binding site present in the variable region of both heavy and light chain polypeptides. These specific regions have been described by: Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of protein of immunological interest. (1991), and Chothia et al., J. Mol. Biol. 196:901-917 (1987) and MacCallum et al., J. Mol. Biol. 262:732-745 (1996), where the definitions, when compared, include overlaps or subsets of amino acid residues. The amino acid residues covering the CDR as defined in each of the references cited above are shown for comparison. Preferably, the term “CDR” is the CDR defined by Kabat based on sequence comparison.

[0044] Table 2: CDR Definition

[0045]

[0046] 1 Residue numbering follows the nomenclature of Kabat et al., as described above.

[0047] 2 Residue numbering follows the nomenclature of Chothia et al., as described above.

[0048] 3 Residue numbering follows the nomenclature of MacCallum et al., as described above.

[0049] As used herein, the term "constant region" refers to the portion of an antibody that is outside the variable domain or variable region. Immunoglobulin light chains have a single-domain "constant region," often referred to as the "CL or CL1 domain." This domain is located at the C-terminus of the VL domain. The constant region of immunoglobulin heavy chains varies depending on the immunoglobulin class (γ, μ, α, δ, ε). Heavy chains γ, α, and δ have a constant region consisting of three immunoglobulin domains (called CH1, CH2, and CH3) and a flexible hinge region separating the CH1 and CH2 domains. Heavy chains μ and ε have a constant region consisting of four domains (CH1 through CH4). The constant domain of the heavy chain is located at the C-terminus of the VH domain.

[0050] The amino acid numbering in the heavy and light chains of immunoglobulins extends from the N-terminus of the Y-configuration branch to the C-terminus at the bottom of each chain. Different numbering schemes are used to define the constant domains of the immunoglobulin heavy and light chains. According to the EU numbering scheme, the constant domains of the IgG heavy chain are identified as follows: CH1—amino acid residues 118 to 215; CH2—amino acid residues 231 to 340; CH3—amino acid residues 341 to 446. According to the Kabat numbering scheme, the constant domains of the IgG heavy chain are identified as follows: CH1—amino acid residues 114 to 223; CH2—amino acid residues 244 to 360; CH3—amino acid residues 361 to 477.

[0051] The “Fc domain” or “Fc region” typically defines a portion of the constant region of the heavy chain, including the CH2 and CH3 domains. The Fc region may also contain residues from the hinge region. The hinge region comprises the portion of the heavy chain molecule that connects the CH1 and CH2 domains. This hinge region contains approximately 25 residues and is flexible, thus allowing the two N-terminal antigen-binding regions to function independently. The hinge region can be subdivided into three distinct domains: upper, middle, and lower hinge regions (Roux KH et al. J. Immunol. 161:4083-90 1998). Antibodies of this invention containing a “fully human” hinge region may comprise one of the hinge region sequences shown in Table 3 below.

[0052] Table 3: Human Hinge Sequence

[0053]

[0054] The Fc region interacts with cell surface receptors called Fc receptors and some proteins in the complement system. This region allows antibodies to activate the immune system, for example, by binding to Fc receptors, and is therefore responsible for mediating antibody effector functions, as defined below.

[0055] Antibody effector functions are an important part of the humoral immune response and form a necessary link between innate and adaptive immunity. Most of these effector functions are induced by the constant (Fc) region of the antibody, which interacts with complement proteins and specific Fc receptors. The term "antibody effector function" is used interchangeably with "Fc effector function" or "Fc region effector function" in this document. The most well-known Fc-mediated antibody effector functions are antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cytophagy (ADCP), and complement-dependent cytotoxicity (CDC). In ADCC, FcγR on the surface of effector cells (natural killer cells, macrophages, monocytes, and eosinophils) binds to the Fc region of IgG that binds to the target cell. This interaction induces the effector cell to release cytotoxic granules (containing perforin and granzymes), leading to the lysis and eventual killing of the target cell.

[0056] The term "fragment"—as used in the context of antibodies of this invention—refers to a portion or part of an antibody or antibody chain containing fewer amino acid residues than a complete or whole antibody or antibody chain. The term "antigen-binding fragment" refers to a polypeptide fragment of an immunoglobulin or antibody that binds to an antigen or competes for antigen binding with a complete antibody (i.e., the complete antibody from which it is derived). The term "fragment" of an antibody molecule, as used herein, includes antigen-binding fragments of antibodies, such as variable domains (VL) of the antibody light chain, variable domains (VH) of the antibody heavy chain, single-chain antibodies (scFv), F(ab')2 fragments, Fab fragments, Fd fragments, Fv fragments, single-arm (monovalent) antibodies, biantibodies, triantibodies, tetraantibodies, or any antigen-binding molecule formed by combining, assembling, or conjugating such antigen-binding fragments. The term "antigen-binding fragment," as used herein, is also intended to encompass antibody fragments selected from unibody, domain antibody, and nanobody. Fragments can be obtained, for example, by chemical or enzymatic treatment of a complete or whole antibody or antibody chain, or by recombinant means.

[0057] As used herein, the term "modified release formulation" refers to a conventional immediate-release formulation that has been modulated to alter the timing and / or rate of release of the active ingredient from the formulation when administered via the same route. This term can encompass a variety of modulated forms, including, but not limited to, slow-release, extended-release, sustained-release, delayed-release, controlled-release, long-acting, or subcutaneous reservoir formulations. For example, extended-release drug products allow for a reduction in dosing frequency compared to drugs presented in immediate-release dosage forms. Delayed-release drug products may release one or more discrete portions of the drug at a later time rather than immediately after administration. Modified release formulations and strategies for altering the timing and / or rate of release of the active ingredient from the formulation are known to those skilled in the art. A popular strategy for developing long-acting systems is to encapsulate the original drug in a hydrogel or large-sized particles that can be injected or implanted subcutaneously to release the load through gradual degradation. An alternative approach is to coat the surface of a subcutaneous reservoir with a masking layer that provides a hydrophilic interface between the tissue fluid and the reservoir, significantly reducing protein adsorption and cell adhesion. There are also many lipid-based platforms for sustained release (such as liposome systems), where the release of drugs encapsulated in liposomes depends on the diffusion of the drug across the lipid bilayer (Rahnfeld & Luciani, 2020; Pharmaceutics; 12(6): 567).

[0058] As used herein, the term "mast cell disease" or "mast cell syndrome" refers to a group of diseases in which mast cell dysfunction plays a major role in the pathogenesis of the disease. Mast cell diseases are conditions in which there is an increase in the number of mast cells, overreaction, or both. Therefore, symptoms are caused by the proliferation and inappropriate activation of excessive mast cells and / or the inappropriate release of mast cell mediators from hypersensitive mast cells. Examples of mast cell diseases include mastocytosis. The symptoms experienced by patients with mast cell diseases are caused by the excessive release of mast cell mediators, most notably histamine. The symptoms experienced depend on the affected organ, such as wheals, skin redness and itching, or gastrointestinal spasms and diarrhea. Sometimes patients experience isolated finding, such as a skin rash associated with mastocytosis; others experience a wide range of symptoms, such as flushing, presyncope, diarrhea, and cramps.

[0059] As used herein, the term “mastocytosis” refers to a mast cell disease typically caused by the clonal proliferation of mast cells, in which these cells abnormally accumulate in tissues including the skin, bone marrow, and gastrointestinal tract.

[0060] As used herein, the term "slow absorption delivery route" refers to a drug delivery route that results in the slow absorption of the active ingredient from the injection site into the patient's bodily fluids (lymph or blood). Intravenous administration is the fastest route of administration because the active ingredient is injected directly into the bloodstream without the need for absorption. Muscle is also rich in blood vessels, and therefore intramuscular administration results in the rapid absorption of the active ingredient through muscle tissue into the bloodstream. As used herein, the term "slow absorption delivery route" may be "slow" relative to intravenous administration and / or relative to intramuscular administration. Subcutaneous tissue has a relatively low vascular density, and therefore, physicians generally consider subcutaneous administration to represent a "slow absorption delivery route" because the active ingredient diffuses through the tissue at a slow, continuous rate. Intradermal administration is classified as another "slow absorption delivery route." Intradermal injection is administered into the dermis (straight beneath the epidermis) and has the longest absorption time of all non-gut routes because this tissue layer has a limited blood supply.

[0061] As used herein, the term "treatment" and its variations refer to therapeutic intervention for a subject with mast cell disease. The treatment methods described herein involve administering anti-mast cell antibodies to a subject via a slow absorption delivery route. Treatment outcomes include improvement or stabilization of the disease, reduction or elimination of disease symptoms, or slowing or eliminating disease progression. Treatment may also include preventative treatment of the disease.

[0062] As used herein, the term "therapeutic effective amount" refers to the amount of anti-mast cell antibody that will elicit the desired biological response (mast cell depletion) in a subject. The amount considered effective can vary depending on the anti-mast cell antibody, the stage and / or severity of the disease, or the age and / or weight of the subject to be treated. A therapeutic effective amount can be readily determined by someone skilled in the art (e.g., the physician treating the subject). A therapeutic effective amount is typically the amount of anti-mast cell antibody that effectively improves or reduces one or more symptoms of mast cell disease.

[0063] As used herein, the term "subject" refers to a mammal, preferably a human. The subject can be male or female. The subject may exhibit one or more symptoms consistent with mast cell disease. In some embodiments, the subject may be a patient, where the patient is an individual receiving medical care and / or actively seeking medical care to treat mast cell disease.

[0064] As used herein, the terms “anaphylactic reaction” or “anaphylactic shock” refer to a severe, potentially life-threatening allergic reaction. Anaphylactic reactions typically occur within minutes of exposure to an allergen, but sometimes can occur up to four hours later. Signs of an anaphylactic reaction include: itchy or raised red rash; swelling of the eyes, lips, hands, and feet; dizziness or fainting; swelling of the mouth, throat, or tongue, which can lead to difficulty breathing and swallowing; wheezing; abdominal pain, nausea, and vomiting; and collapse and unconsciousness. Anaphylactic reactions occur rapidly and are systemic, affecting one or more organ systems, typically in areas with a relative abundance of mast cells. The pathophysiological basis of anaphylactic reactions is exposure to allergens or other factors that activate mast cells or basophils, thereby promoting degranulation and the immediate (5 to 30 minutes) release of pre-formed mediators (histamine, trypsin, carboxypeptidase A, and proteoglycans), the synthesis of arachidonic acid metabolites (prostaglandins, leukotrienes) and platelet-activating factor (PAF), and the delayed (2 to 6 hours) production of cytokines (TNF-α) and chemokines due to increased gene expression. These mediators are considered to be the cause of symptoms associated with anaphylactic reactions. The most widely used models for studying anaphylactic responses are passive cutaneous anaphylaxis (PCA) and passive systemic anaphylaxis (PSA), which can be examined in genetically modified mouse strains.

[0065] B. Methods to deplete mast cells & methods to treat mast cell diseases

[0066] In a first aspect, the present invention relates to a method for depleting mast cells in a subject. Many pathological conditions are characterized by the presence of abnormal mast cells or the accumulation of an excessive number of mast cells. Therefore, one object of the present invention is to deplete the number of mast cells in a subject where this is desired.

[0067] In another aspect, the present invention provides a method for treating mast cell disorders in subjects with this need. Typical characteristics of subjects with this need are the presence of abnormal mast cells and / or the accumulation of an excessive number of mast cells.

[0068] The method of the present invention includes administering an effective amount of anti-mast cell antibody to a subject, wherein the anti-mast cell antibody is administered via a slow absorption delivery route. As reported elsewhere herein, administration of anti-mast cell antibody via a slow absorption delivery route allows for effective depletion of mast cells while avoiding anaphylactic reactions that may occur after administration of anti-mast cell antibody via other routes, such as intravenous administration.

[0069] In some embodiments, the method depletes the number of mast cells in a subject. In some embodiments, the method provided herein depletes mast cells in one or more of the following tissues or organs: skin; mesentery; lung; small intestine; and large intestine. As used herein, the term "depleted mast cells" means a reduction in the number of mast cells present in the subject after administration of an anti-mast cell antibody compared to the number of mast cells present in the subject prior to administration of the anti-mast cell antibody. In some embodiments, "depleted mast cells" means that the number of mast cells present in the subject is reduced to below a predetermined threshold after administration of the anti-mast cell antibody.

[0070] Technicians are familiar with appropriate methods for assessing the number of mast cells in a subject. The number of mast cells can be determined using histochemical methods. For example, tissue samples from the subject can be biopsied, fixed in formalin, and embedded in paraffin. The sections can then be stained with toluidine blue, methylene blue, or Csaba staining agents, which bind to heparin and other acidic mucopolysaccharides contained in mast cells, causing them to exhibit metachromatic staining. Alternatively, the total number of mast cells can be estimated based on serum plasma trypsin levels (a marker of mast cell load). Mast cells release trypsin in response to external stimuli, but they also release small amounts of trypsin under steady-state conditions, resulting in a correlation between circulating trypsin levels and the number of mast cells in the tissue.

[0071] Application route

[0072] As illustrated in the examples herein, mast cells can be safely and effectively depleted by administering anti-mast cell antibodies according to an optimized delivery route. In particular, the method of the present invention requires the administration of anti-mast cell antibodies to the subject via a slow absorption delivery route. The term "slow absorption delivery route" is defined elsewhere herein and generally refers to administration of a drug via a route slower than intravenous administration (i.e., where the drug enters the circulation directly). Delivery routes considered in the art as "slow absorption delivery routes" include, but are not limited to, subcutaneous and intradermal administration. These administration routes involve delivery to tissues with limited blood supply, and therefore the drug must diffuse from the injection site through the tissue to reach the bloodstream. This results in a "slow absorption delivery route," for example, compared to intravenous administration.

[0073] Therefore, in some embodiments of the first and second aspects of the invention, anti-mast cell antibodies may be administered via any delivery route known to those skilled in the art that achieves slow absorption of the antibody. In some embodiments, anti-mast cell antibodies are administered subcutaneously. In some embodiments, anti-mast cell antibodies are administered via a subcutaneous implant. In some embodiments, anti-mast cell antibodies are administered intradermally. By the same reasoning, in all embodiments of the first and second aspects of the invention, anti-mast cell antibodies are not administered to the subject via delivery routes known to those skilled in the art that achieve rapid and immediate targeting of mast cells. In some embodiments, anti-mast cell antibodies are not administered intravenously.

[0074] This article provides a method for depleting mast cells in a subject, the method comprising administering an effective amount of an anti-mast cell antibody to the subject, wherein the anti-mast cell antibody is administered subcutaneously to the subject.

[0075] This article provides a method for depleting mast cells in a subject, the method comprising administering an effective amount of an anti-mast cell antibody to the subject, wherein the anti-mast cell antibody is administered to the subject via a subcutaneous implant.

[0076] This article provides a method for depleting mast cells in a subject, the method comprising administering an effective amount of an anti-mast cell antibody to the subject, wherein the anti-mast cell antibody is administered intradermally to the subject.

[0077] This article provides a method for depleting mast cells in a subject, the method comprising administering an effective amount of an anti-mast cell antibody to the subject, wherein the anti-mast cell antibody is not administered intravenously.

[0078] This document provides a method for depleting mast cells in a subject, the method comprising administering an effective amount of an anti-mast cell antibody to the subject, wherein the anti-mast cell antibody is administered to the subject via a slow absorption delivery route, and wherein the anti-mast cell antibody is not administered intravenously.

[0079] This article provides a method for treating mast cell disease in subjects with this need, the method comprising administering a therapeutically effective amount of an anti-mast cell antibody to the subject, wherein the anti-mast cell antibody is administered subcutaneously to the subject.

[0080] This article provides a method for treating mast cell disease in subjects with this need, the method comprising administering a therapeutically effective amount of an anti-mast cell antibody to the subject, wherein the anti-mast cell antibody is administered to the subject via a subcutaneous implant.

[0081] This article provides a method for treating mast cell disease in subjects with this need, the method comprising administering a therapeutically effective amount of an anti-mast cell antibody to the subject, wherein the anti-mast cell antibody is administered intradermally to the subject.

[0082] This article provides a method for treating mast cell disease in subjects with this need, the method comprising administering a therapeutically effective amount of an antimast cell antibody to the subject, wherein the antimast cell antibody is administered to the subject without intravenous administration.

[0083] This article provides a method for treating mast cell disease in subjects with this need, the method comprising administering a therapeutically effective amount of an antimast cell antibody to the subject, wherein the antimast cell antibody is administered to the subject via a slow absorption delivery route, and wherein the antimast cell antibody is not administered intravenously.

[0084] T max The maximum drug concentration (C) is reached in the plasma after administration of the drug. max The time spent. Generally speaking, compared with administration via known rapid delivery routes (e.g., intravenous administration), slow absorption delivery routes will result in T cells of anti-mast cell antibodies being released. max Increase. In some embodiments, the anti-mast cell antibody is administered to the subject via a slow absorption delivery route, thereby increasing the T-cell activity of the anti-mast cell antibody. max T cells with the same anti-mast cell antibody as those administered intravenously max This represents an increase compared to previous methods. In some embodiments, the anti-mast cell antibody is administered to the subject via a slow absorption delivery route, resulting in a higher Tg of the anti-mast cell antibody. max T cells with the same anti-mast cell antibody as those administered intravenously max Compared to an increase of at least 10%, at least 15%, at least 20%, at least 25%, or at least 30%.

[0085] In some embodiments, the methods described herein reduce or prevent mast cell activation. In some embodiments, the methods reduce or prevent mast cell degranulation. In some embodiments, the methods reduce or prevent allergic reactions. In some embodiments, the methods reduce or prevent anaphylactic reactions. In some embodiments, the methods reduce the severity of allergic reactions. In some embodiments, the methods reduce the severity of anaphylactic reactions.

[0086] In some embodiments, the administration of anti-mast cell antibodies does not induce mast cell activation. In some embodiments, the administration of anti-mast cell antibodies does not induce mast cell degranulation. In some embodiments, the administration of anti-mast cell antibodies does not induce an allergic reaction. In some embodiments, the administration of anti-mast cell antibodies does not induce a hypersensitivity reaction.

[0087] Anti-mast cell antibodies

[0088] The anti-mast cell antibody applied according to the method described herein needs to bind to mast cells / initiate an immune response. More specifically, the anti-mast cell antibody preferentially binds to proteins (e.g., receptors or transmembrane proteins) expressed on the surface of mast cells. Preferably, the target protein is uniquely expressed on mast cells, or at least more abundantly expressed on mast cells than on any other cell type. In this way, the method of the present invention can specifically target mast cells for exhaustion.

[0089] In some embodiments, the anti-mast cell antibody specifically binds to proteins expressed on the surface of mast cells. In some embodiments, the anti-mast cell antibody specifically binds to mast cell-specific surface proteins. In some embodiments, the anti-mast cell antibody specifically binds to receptors expressed on the surface of mast cells. In some embodiments, the anti-mast cell antibody specifically binds to mast cell-specific receptors. In some embodiments, the receptor is c-KIT. In some embodiments, the anti-mast cell antibody is an anti-c-KIT antibody.

[0090] The term "antibody" is defined elsewhere herein and, in the context of this invention, is used relatively broadly to refer to any antigen-binding molecule that has binding specificity to mast cell targets. The term "antibody" as used herein is broad enough to encompass antigen-binding fragments as defined elsewhere herein. The term "antibody" also encompasses, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and VHH antibodies, provided they exhibit appropriate immunospecificity against mast cell proteins.

[0091] As used herein, the term "monoclonal antibody" refers to an antibody derived from a population of substantially homogeneous antibodies; that is, the individual antibodies constituting this population are identical, except that they may be present in small amounts as naturally occurring mutations. Monoclonal antibodies are highly specific, targeting a single antigenic site. Furthermore, unlike conventional (polyclonal) antibody formulations, which typically contain different antibodies targeting different determinants (epitopes) on an antigen, each monoclonal antibody targets a single determinant or epitope on the antigen.

[0092] An "antibody fragment" or "antigen-binding fragment" comprises a portion of a full-length antibody, typically containing its antigen-binding domain or variable domain. Antibody fragments are described elsewhere in this document, and some examples of antibody fragments include Fab, Fab', F(ab')2, bispecific Fab' and Fv fragments, biantibodies, linear antibodies, single-chain antibody molecules, single-chain variable fragments (scFv), and multispecific antibodies formed from antibody fragments (see Holliger and Hudson, Nature Biotechnol. 23:1126-36 (2005), the contents of which are incorporated herein by reference).

[0093] The anti-mast cell antibodies used according to the methods described herein are intended for human therapeutic use and will therefore typically be immunoglobulins of the IgA, IgD, IgE, IgG, or IgM type, typically of the IgG type, in which case they may belong to any of the four subclasses IgG1, IgG2a and b, IgG3, or IgG4. In some preferred embodiments, the anti-mast cell antibody is an IgG antibody. IgG1 antibodies are particularly preferred. Monoclonal antibodies are preferred because they are highly specific, targeting a single antigenic site.

[0094] Anti-mast cell antibodies can exhibit high human homology. Such antibodies with high human homology may comprise antibodies containing the VH and VL domains of a naturally occurring non-human antibody, exhibiting a sufficiently high percentage of sequence identity with human germline sequences. In some embodiments, the antibody or its antigen-binding fragment is a humanized or germline variant of a non-human antibody.

[0095] The anti-mast cell antibody used according to the method described herein may comprise or consist of one or more antibody variable domains linked to an Fc region (optionally linked via an antibody hinge region). In some embodiments, the hinge region and / or Fc region have a fully or substantially human amino acid sequence.

[0096] In the context of this invention, it is particularly preferred that anti-mast cell antibodies can participate in Fc-mediated effector functions to induce effective mast cell exhaustion. Some examples herein demonstrate that anti-mast cell antibodies with a functional Fc region are more effective at exhausting mast cells than anti-mast cell antibodies with a loss-of-function Fc region. Furthermore, data show that anti-mast cell antibodies with an Fc region modified to enhance antibody-dependent cytotoxicity (ADCC) activity are particularly effective in mast cell exhaustion.

[0097] Therefore, in some embodiments, the anti-mast cell antibody comprises an Fc region having one or more Fc effector functions. In some embodiments, the Fc region is modified to enhance one or more Fc effector functions. In some embodiments, the Fc region has enhanced Fc effector function relative to a wild-type Fc region (particularly a wild-type human Fc region). In some embodiments, the Fc region has enhanced Fc effector function relative to a wild-type human IgG Fc region (particularly a wild-type human IgG1 Fc region).

[0098] In some embodiments, one or more Fc effector functions are selected from antibody-dependent cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC). In some preferred embodiments, the Fc region has antibody-dependent cytotoxicity (ADCC) or is modified to enhance antibody-dependent cytotoxicity (ADCC).

[0099] In some embodiments, the anti-mast cell antibody is a human IgG antibody. In some embodiments, the anti-mast cell antibody is a human IgG1 antibody. In other embodiments, the anti-mast cell antibody is a human IgG2 antibody.

[0100] The anti-mast cell antibody used in the method according to the invention can be modified by any suitable alteration and / or post-translational modification of the amino acid sequence known to those skilled in the art to enhance Fc effector function, particularly to enhance ADCC. Alterations to the amino acid sequence of the Fc region to enhance Fc effector function may include amino acid substitutions, deletions, and / or insertions. For example, mutations that improve the binding affinity of Fc to specific FcγR can be introduced. In some embodiments, the Fc region of the anti-mast cell antibody contains the amino acid substitution S239D / I332E.

[0101] Fucose-deficient antibodies have shown 50-fold higher binding to FcγRIIIa and enhanced ADCC activity. Several strategies can be employed to control Fc glycosylation levels during upstream preparation, including chemical inhibitors, culture process parameters, culture medium supplements, and cell line engineering. These strategies are known to those skilled in the art. Therefore, in some embodiments, the Fc region of the anti-mast cell antibody is non-fucosylated. Terminal galactosylation of recombinant IgG (i.e., galactose glycosylation) has not been shown to affect antibody-antigen binding; however, the presence of galactose has been reported to lead to increased CDC activity in some IgGs. Therefore, in some embodiments, the Fc region of the anti-mast cell antibody is galactosylated.

[0102] In certain embodiments where the Fc region of an anti-mast cell antibody is modified to enhance the function of one or more effectors, the anti-mast cell antibody exhibits increased binding affinity for FcγR compared to unmodified antibodies. In certain embodiments where the Fc region of an anti-mast cell antibody is modified to enhance the function of one or more effectors, the anti-mast cell antibody exhibits increased binding affinity for FcγRIIIa (CD16) compared to unmodified antibodies.

[0103] mast cell disease

[0104] This invention provides a method for treating mast cell disease in subjects with this need. The invention also provides an anti-mast cell antibody used in the method for treating mast cell disease. Additionally, the invention provides the use of the anti-mast cell antibody in the preparation of a medicament for treating mast cell disease. In all cases, the method of treatment and use includes administering a therapeutically effective amount of the anti-mast cell antibody to a subject, wherein the anti-mast cell antibody is administered to the subject via a slow absorption delivery route.

[0105] The term "mast cell disease" is defined elsewhere in this document. In some embodiments, the mast cell disease to be treated according to the treatment methods described herein is mastocytosis.

[0106] In some embodiments of the invention, the subject is a human being. In some embodiments, the subject has an abnormally high mast cell count. In some embodiments, the subject has a high mast cell load. Mast cell counts in the skin are typically confirmed by performing a biopsy, in which a small skin sample is taken and examined for mast cells. Other tests that can be used to confirm systemic mast cell counts include measuring blood trypsin levels.

[0107] The anti-mast cell antibody used in the method according to the invention can be administered according to a suitable dosing regimen. Particularly preferred is that the anti-mast cell antibody is administered to the subject in accordance with the number of mast cells required for effective depletion. Therefore, in some embodiments, the method includes administering one or more doses of the anti-mast cell antibody to the subject. In some embodiments, the method includes administering at least two doses of the anti-mast cell antibody. In some embodiments, the method includes administering at least three doses of the anti-mast cell antibody. In some embodiments, the anti-mast cell antibody is administered to the subject daily. In some embodiments, the anti-mast cell antibody is administered to the subject every two days. In some embodiments, one or more doses of the anti-mast cell antibody are administered to the subject, wherein one or more doses are administered to the subject every two days. In some embodiments, the anti-mast cell antibody is administered to the subject every seven days. In some embodiments, one or more doses of the anti-mast cell antibody are administered to the subject, wherein one or more doses are administered to the subject every seven days.

[0108] Pharmaceutical Composition

[0109] The anti-mast cell antibodies used in the methods described herein can be formulated into pharmaceutical compositions for administration to a subject. Therefore, in some embodiments, the anti-mast cell antibodies are formulated into pharmaceutical compositions.

[0110] Pharmaceutical compositions can be formulated with pharmaceutically acceptable carriers or diluents, as well as any other known excipients and excipients, using conventional techniques, such as those disclosed in Remington: The Science and Practice of Pharmacy, 19th edition, edited by Gennaro, Mack Publishing Co., Easton, Pa., 1995. The term "pharmaceutically acceptable carrier" refers to a carrier or excipient that is inherently non-toxic. Some examples of such excipients are, but are not limited to, saline, Ringer's solution, dextrose solution, and Hanks' solution. Non-aqueous excipients, such as non-volatile oils and ethyl oleate, may also be used.

[0111] Under the conditions of preparation and storage, pharmaceutical compositions must generally be sterile and stable. The composition may be formulated as a solution, microemulsion, liposome, or other ordered structure suitable for high drug concentrations. Some examples of suitable aqueous and non-aqueous carriers that can be used for said pharmaceutical compositions include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof; vegetable oils (e.g., olive oil); and injectable organic esters (e.g., ethyl oleate). Appropriate flowability can be maintained, for example, by using coating materials (e.g., lecithin), by maintaining the desired particle size in the case of dispersions, and by using surfactants.

[0112] The pharmaceutical composition may also contain excipients such as preservatives, wetting agents, emulsifiers, and dispersants. The presence of microorganisms can be prevented by sterilization procedures and by including a variety of antibacterial and antifungal agents, such as parabens, chlorobutanol, phenols, sorbic acid, etc. It is also desirable to include isotonic agents in the composition, such as sugars, polyols such as mannitol, sorbitol, glycerol, or sodium chloride. Pharmaceutically available antioxidants may also be included, such as (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants, such as ascorbate palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and (3) metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0113] According to the present invention, the pharmaceutical composition containing anti-mast cell antibodies must be administered to the subject via a slow absorption delivery route. For example, administration may be parenteral, preferably by subcutaneous or intradermal injection.

[0114] In addition to optimizing the route of drug administration, the pharmaceutical composition may be formulated to further slow the rate of drug absorption. A release-modified formulation, as defined elsewhere herein, refers to a pharmaceutical formulation that thereby alters the time and / or rate of drug release from the formulation. In some embodiments, the pharmaceutical composition is formulated for the slow absorption of anti-mast cell antibodies. In some embodiments, the pharmaceutical composition is a release-modified formulation. In some embodiments, the pharmaceutical composition is a release-modified formulation selected from: slow-release, prolonged-release, long-acting-release, sustained-release, delayed-release, or controlled-release.

[0115] The invention will now be further understood with reference to the following non-limiting examples.

[0116] Example

[0117] The following examples demonstrate the ability of anti-mast cell antibodies to achieve safe and effective mast cell depletion. Results show that anti-mast cell antibodies with Fc effector function can be administered without inducing anaphylactic reactions, particularly via subcutaneous injection. Effective mast cell depletion and a significant reduction in mast cell-mediated inflammatory responses can be achieved when the anti-mast cell antibodies illustrated below are administered subcutaneously. Importantly, antibody-mediated mast cell depletion successfully reduces the severity of anaphylactic reactions, as illustrated using a passive systemic anaphylactic reaction model.

[0118] Example 1: Mast cell exhaustion using an IgG antibody targeting c-KIT

[0119] It is well known that mast cells are difficult to target with antibodies due to their ability to degranulate and induce anaphylactic reactions. However, it was found that intraperitoneal injection of a modified mouse IgG2a anti-c-KIT monoclonal antibody—clone ACK2—with enhanced ADCC activity (ADCC+) induced hypothermia (a sign of anaphylactic reaction) in mice.

[0120] In the first experiment designed to address this problem, a “desensitization” approach was employed, similar to that previously reported using anti-FcεRI antibodies (Finkelman et al., 2020). It was hypothesized that mast cell activation could be prevented by dose-escalating anti-c-KIT ADCC+ mAb. Mice were administered intraperitoneal injections of escalating doses of anti-c-KIT ADCC+ antibody, an isotype control, or a carrier (PBS) every 30 minutes at doses of 0.1, 0.3, 1, 3, and 10 µg / injection. Mouse temperature was monitored; see [link to relevant documentation]. Figure 1 A.

[0121] During the desensitization phase, mice exhibited signs of anaphylactic shock once a dose of 3 µg was reached. This indicates that repeated systemic antibody injections eventually reached a dose capable of activating mast cells.

[0122] After the desensitization phase, the same mouse was challenged again with a larger concentration of 30 µg of anti-c-KIT ADCC+ mAb. Figure 1 B, left image (day 1). This desensitization with anti-c-KIT ADCC+ mAb showed effectiveness, as there was no shock compared to mice treated with the isotype or PBS control during the desensitization phase, but the isotype showed reduced shock compared to the PBS control. This is likely due to the blockade of FcγR. Subsequent treatment after concentrated administration did not induce shock ( Figure 1 (Figure B, middle image (day 3) and right image (day 5)). This further suggests that this may be a safe method. However, given the significant shock that still occurred during the initial desensitization phase, alternative methods need to be considered.

[0123] On day 7, mast cells in different groups were quantified to determine the degree of exhaustion caused by the desensitization method. Figure 1 C). Effective depletion of mast cells in the peritoneal cavity was observed. Interestingly, the same depletion was observed in all groups receiving at least three injections of 30 µg anti-c-KIT ADCC+ mAb, indicating that effective depletion can be achieved with this mAb. Given the positive indications from the desensitization approach, it is speculated that slower diffusion may be beneficial in improving the safety profile.

[0124] Example 2: Effects of different administration routes of anti-c-KIT antibody

[0125] Mouse IgG2a anti-c-KIT monoclonal antibody clone ACK2 (which is commercially available) is produced in different Fc backbone forms: loss-of-function (no effector function); wild-type; and ADCC+ (modified to have enhanced ADCC function). The ADCC+ form contains the amino acid substitution S239D / I332E in the Fc region.

[0126] like Figure 2 As shown, intraperitoneal injection into wild-type mice ( Figure 2 B) Intravenous ( Figure 2 C), or subcutaneous ( Figure 2 D) Inject 30 µg of Fc-deficient or ADCC+ mAb. Subcutaneous experiments also included wild-type ACK2 and isotype controls.

[0127] Fc-deficient mAbs did not induce hypothermia under any circumstances. Results showed that intravenous injection of anti-c-KIT ADCC+ mAbs induced the fastest anaphylactic response, while intraperitoneal injection showed a slightly delayed but similar response. Most interestingly, subcutaneous injection of any of the following antibodies did not induce any hypothermia: isotype control, anti-c-KIT Fc-deficient, WT, or ADCC+. This is likely due to the slower diffusion of antibodies through subcutaneous tissue.

[0128] One hour after antibody challenge, blood samples were collected and serum was separated, which was subsequently used to quantify the release of mast cell-specific proteases MCPT-1 (produced by mucosal mast cells) and MCPT-6 (produced by connective tissue mast cells). Consistent with the hypothermia results, the data showed that MCPT-6 was released at high levels upon intravenous or intraperitoneal injection of anti-c-KIT ADCC+, but not upon injection of Fc loss-of-function antibodies. Subcutaneous injection of wild-type and anti-c-KIT ADCC+ antibodies did not increase MCPT-6 levels or increased very slightly; however, this was not in the same range as with other injection routes. No detectable MCPT-6 was found in the isotype control and the anti-c-KIT Fc loss-of-function antibody treatment groups. Regardless of the injection route, MCPT-1 levels were undetectable in almost all samples, indicating that mucosal mast cells (the only mast cells expressing MPCT-1) were not activated by this treatment. These data confirm the reliable safety profile of anti-c-KIT antibodies with Fc effector function upon subcutaneous injection.

[0129] Figure 3 This demonstrates the rapid onset of allergic reactions following intraperitoneal injection of 30 µg of anti-c-KIT wild-type and anti-c-KIT ADCC+. Figure 3 C). The percentage of mast cells present in the peritoneal cavity was also assessed on day 7 after administration. Data showed that all forms of anti-c-KIT antibodies reduced mast cell number upon intraperitoneal administration, with anti-c-KIT ADCC+ being the most effective, followed by anti-c-KIT wild-type, and then anti-c-KIT Fc loss-of-function type. Figure 3 B).

[0130] Example 3: Subcutaneous injection of anti-c-KIT antibody (ACK2 ADCC+) can deplete mast cells without inducing allergic reactions. reaction

[0131] In Example 3, C57BL / 6 wild-type mice were subcutaneously injected with anti-c-KIT antibody three times within one week (days 0, 2, and 4), followed by mast cell quantification on day 7. Figure 4 A). For example Figure 4As shown in B, subcutaneous administration of anti-c-KIT antibodies did not induce an allergic reaction, regardless of the antibody form. Compared to the control, this 3-dose administration regimen resulted in effective depletion of mast cells in the peritoneal cavity with all three anti-c-KIT antibody forms (Fc loss-of-function, wild-type, and ADCC+), with the most significant reduction observed in ADCC+ antibodies (see [link to B]). Figure 4 C). The same trend in mast cell quantification was observed in dorsal skin and mesenteric windows (see [link]). Figure 4 D and 4F). No significant mast cell depletion was observed in the ear skin (see D and 4F). Figure 4 E); however, this is not unexpected, given the large mast cell load in the middle ear of mice (about 3 times that of the skin on the back), and the depletion in this tissue would likely require a much longer treatment protocol.

[0132] Alternative treatment regimens were investigated, which included two subcutaneous injections one week apart, allowing for a longer period of time to deplete mast cells. Figure 5 A). For example Figure 5 As can be seen in B, 30 µg is the optimal dose to deplete approximately 90% of the mast cells in the peritoneal cavity.

[0133] Example 4: Efficacy of subcutaneous injection of anti-c-KIT antibody on mast cell number in different organs

[0134] follow Figure 6 The protocol described in A allows for parallel testing of different anti-c-KIT antibody forms and analysis of mast cell numbers in various tissues. Figure 6 B and 6C show the mast cell profile in the peritoneal cavity. Significantly greater exhaustion of absolute mast cell numbers was observed after administration of anti-c-KIT ADCC+ antibody compared to the isotype control and the Fc loss-of-function treatment group.

[0135] like Figure 6Histological analysis was also performed, as shown in Figure D. Mesenteric windows were stained with Csaba stain, and the results showed that the ADCC+ form was significantly more effective in depletion than the isotype control and other forms of anti-c-KIT mAb. Staining of back and ear skin with toluidine blue indicated that the ADCC+ form remained consistently effective in depletion compared to the isotype control in both tissue samples. In back skin sections, the anti-c-KIT ADCC+ antibody was also more effective than the Fc-deficient form, and tended to be significantly more effective than the WT form in ear skin (p=0.0603). Lung sections were also analyzed by toluidine blue staining; however, this only stained heparin-positive mast cells around the bronchi. This resulted in variability in mast cell number based on the sections analyzed. Finally, mast cells in small and large intestine sections were analyzed by MCPT-1 immunofluorescence staining. This also clearly showed that mast cells in both intestinal tissues were significantly depleted by the anti-c-KIT ADCC+ antibody compared to the control group. In the WT treatment group, mast cells in the small intestine were also depleted, but not as significantly as in the ADCC+ antibody treatment group.

[0136] Example 5: Effects of anti-c-KIT mAb on leukocyte populations and bone marrow progenitor cells

[0137] Given that many common progenitor cells of leukocytes express the c-KIT receptor, a whole-leukocyte cohort was run to examine the effect of treatment on the major immune population in the blood. Figure 7 This is an important safety aspect of targeting c-KIT, because treatments in clinical settings, particularly tyrosine kinase inhibitors (TKIs), often result in immunosuppression due to global, nonspecific inhibition of tyrosine kinase receptors.

[0138] Blood samples were taken on day 13 (the day before sacrifice), and 200 µl of blood was quantitatively analyzed for FACS using counting beads. No significant changes in the absolute number of major lymphocytes (B cells, T cells, and NK cells) were observed (see [link to relevant documentation]). Figure 7 F, 7G, and 7H). This indicates that a fully functional adaptive immune system remains regardless of the antibody form. However, when examining cell populations from myeloid origin, significant changes were observed after treatment with WT and ADCC+ anti-cKIT mAb, but not with the Fc loss-of-function form (see Fc). Figure 7 (A through E). WT treatment resulted in a significant reduction in the following myeloid populations: monocytes, neutrophils, dendritic cells, basophils, and eosinophils. Similarly, ADCC+ antibody treatment resulted in a reduction in these same myeloid cell populations, except for neutrophils, which was reduced to a less significant degree. Therefore, these reductions appear to be dependent on Fc effector function.

[0139] Since bone marrow progenitor cells also express the c-KIT receptor, the progenitor cell population on day 14 of the exhaustion protocol was examined. Following the... Figure 6 The same treatment protocol shown in A, measuring Lin from bone marrow. - Sca-1 + Kit + (LSK), Lin - Kit + (LK) and Lin - Sca-1 + Kit - (LSK-) cell levels ( Figure 8 No significant differences were observed between the control and all treatment groups. This indicates a favorable safety profile, as it implies potentially less immunosuppression, especially compared to current treatments such as imatinib or avapritinib, which are small-molecule TKIs that inhibit c-KIT and other receptors. These results also suggest that the effects observed in blood myeloid assays are more likely due to potential depletion of circulating progenitor cells or tissue recruitment.

[0140] Example 6: The efficacy of mast cell depletion in reducing allergic reactions

[0141] To observe the effect of anti-c-KIT antibody treatment on mast cell-mediated responses, two different IgE-induced passive systemic anaphylactic response protocols were performed after administration of mast cell depletion antibodies.

[0142] In one study, mast cell depletion was performed by IgE sensitization on day 7, followed by antigen challenge on day 8. Figure 9 As shown in the second study, mast cell depletion was performed by IgE sensitization on day 14, followed by antigen challenge on day 15, as illustrated. Figure 10 As shown in A.

[0143] In both studies, core body temperature was monitored one hour after challenge to examine for hypothermia, the gold standard for anaphylactic reactions in mice. Control mice showed a significant temperature drop, averaging about 5°C. Mice treated with Fc loss-of-function anti-c-KIT antibodies showed some protection against this temperature drop. However, hypothermia was significantly less severe in the WT and ADCC+ anti-c-KIT antibody treatment groups (see [link to study]). Figure 9 and Figure 10 B). Although no clinical scoring was performed, significant behavioral differences were observed: mice treated with WT and ADCC+ antibodies behaved normally after the attack, while the isotype and Fc loss-of-function groups exhibited scratching, discomfort, and prone posture.

[0144] In the second study, blood was collected 60 minutes after the attack for serum analysis of mast cell-specific proteases MCPT-1 and MCPT-6, which were subsequently quantified by ELISA. Figure 10 (C) Serum analysis showed that the ADCC+ form most significantly reduced serum MCPT-1 levels compared to the isotype control, but the WT treatment group also showed a significant reduction in MCPT-1 levels. The same trend was observed when examining MCPT-6 levels, with both the WT and ADCC+ treatment groups showing significant reductions in serum levels of this protease. In both the WT and ADCC+ treatment groups, both MCPT-1 and MCPT-6 were reduced to an average of one-tenth, and were completely undetectable in some samples. In the Fc loss-of-function treatment group, some samples also showed undetectable levels of the protease; however, in most samples, levels remained similar to those in the isotype control group.

[0145] Most promisingly, ADCC+ antibody treatment significantly reduced MCPT-1 compared to the Fc loss-of-function treatment group. This further demonstrates that the ADCC+ antibody form is superior to the Fc loss-of-function form in depleting mast cells, thus providing better protection against IgE-mediated allergic reactions, and offering additional evidence that antibodies without Fc effector function (e.g., bazolizumab) are less effective against invasive mast cell-mediated diseases.

[0146] To further test the protective effect of mast cell exhaustion, additional mast cell-dependent responses were investigated. In this further study, the ciprofloxacin-induced passive systemic anaphylactic response was evaluated. Figure 11 A). Ciprofloxacin-induced passive systemic anaphylaxis is known to be a mast cell and mrgprb2 (a mouse ortholog of MRGPRX2)-dependent response, because in this PSA model, mrgprb2 KO mice are protected from signs of anaphylaxis. Mast cell activation induced by mrgprb2 engagement is termed a pseudoallergic reaction, i.e., not an IgE-induced response. It is significantly less severe in terms of hypothermia, as evidenced by the approximately 1.5°C temperature drop in controls. Figure 11 B). This experiment showed that Fc loss-of-function treatment significantly reduced severe hypothermia, while ADCC+ treatment provided complete protection against hypothermia.

[0147] Serum was collected 60 minutes after the attack, and MCPT-1 and MCPT-6 ELISA were performed as markers of mast cell activation. Figure 11C). MCPT-1 was not detected in any treatment group. This is because ciprofloxacin only activates mast cells expressing the mrgprb2 receptor, which is limited to connective tissue mast cells that do not express MCPT-1. However, MCPT-6 release was observed in the isotype treatment group. Serum MCPT-6 was significantly reduced in both the Fc loss-of-function and ADCC+ antibody treatment groups, and more promisingly, the reduction in MCPT-6 levels was even more significant in the ADCC+ treatment group.

[0148] Materials and methods

[0149] Table 5: Antibodies

[0150]

[0151]

[0152] mice

[0153] Wild-type 7-week-old C57BL / 6 mice were obtained from Charles River Laboratories and housed at the CREFRE animal facility (Toulouse, Purpan). Mice were used from 8 weeks of age onwards unless otherwise stated. All experiments required approval from the local ethics committee.

[0154] Anti-c-Kit mAb treatment

[0155] When testing the safety profile, administer 30 µg / 100 µl of different forms (Fc loss-of-function, WT, or ADCC+) of anti-mouse c-KIT (CD117) mAb (clone ACK2) or an isotype control (ADCC+) subcutaneously (sc), intraperitoneally (ip), or intravenously (iv). In all other experiments, unless otherwise specified, administer 30 µg / 100 µl of these antibodies subcutaneously.

[0156] The ACK2 monoclonal antibody reacted with mouse CD117 (also known as the c-KIT receptor). ACK2 has been reported to block c-KIT function. Motavizumab is an antibody against respiratory syncytial virus (RSV) and was used as an isotype control.

[0157] IgE passive systemic anaphylactic reaction (PSA)

[0158] Mice were sensitized with 10 μg / 200 μl IgE against DNP (clone SPE-7). Twenty-four hours later, the mice were challenged by intravenous injection of 500 μg / 200 μl DNP-I (Sigma). The anaphylactic reaction was observed as a decrease in body temperature (measured rectally) for one hour.

[0159] tissue samples

[0160] Tissue samples (skin from the back, ears, lungs, small intestine, and large intestine) were harvested and placed in formaldehyde (4% w / v) at room temperature for 24 hours. Afterward, the samples were placed in 70% EtOH at 4°C until contained in paraffin blocks. Sections were cut to a thickness of 5 μm.

[0161] Toluidine Blue

[0162] In short, the sections were dewaxed using xylene and ethanol in decreasing concentrations. Subsequently, the sections were stained with toluidine blue (Sigma) for 30 seconds and then rinsed with deionized water. The slides were mounted and scanned using a slide scanner (Panoramic 250). Mast cells were quantified using a case viewer and expressed as mast cell number / mm². 2 express.

[0163] Csaba staining

[0164] Mesenteric window directly harvested from Superfrost TM Plus, adhere the slide to a microscope slide, allow it to dry, and then fix it in Carnoy fixative (3 EtOH: 2 chloroform: 1 acetic acid). Remove excess tissue (e.g., intestinal / adipose tissue), and stain the sample in Csaba at room temperature for 15 minutes, followed by washing in deionized water and dehydration with xylene. Mount the slide and image it by mast cell number / mm². 2 Quantitative.

[0165] Immunofluorescence (MCPT-1 staining)

[0166] In short, sections were dewaxed using xylene and ethanol at decreasing concentrations. Subsequently, sections were subjected to antigen retrieval in citrate buffer at 95°C for 20 minutes and then cooled in the buffer for 20–30 minutes. Next, sections were blocked with 1% BSA PBS at room temperature for 2 hours. After this, sections were washed with PBS and stained overnight at 4°C with primary antibody rat anti-mMCPT1 (clone RF6.1) (final concentration: 2.5 ug / ml). The next day, primary antibody was stained for 2 hours at room temperature with goat anti-rat secondary antibody (AF657). Slides were washed and mounted with Dap-prolong gold to visualize the entire tissue section. Images were acquired using a Zeiss confocal microscope and / or by scanning with a slide scanner (panoramic 250).

[0167] Serum collection and ELISA

[0168] Collect blood at the endpoint. Allow the blood to clot at room temperature for 30 minutes, then rotate the sample at 10,000 × g for 10 minutes. Collect the serum and store it at -20°C until analysis.

[0169] Perform the MCPT-1 ELISA (Thermofisher, BMS6005) according to the manufacturer's instructions. Use serum diluted 1:50 in assay buffer.

[0170] Perform the MCPT-6 ELISA (Thermofisher, #EM51RB) according to the manufacturer's instructions. Use serum diluted 1:2 in assay buffer.

[0171] Flow cytometry

[0172] Flow cytometry was performed on a MACQuant10 (Miltenyi) or FORTESA (BD Bioscience). Analyzing was performed using FlowJo (TreeStar).

[0173] blood

[0174] Blood samples were harvested in 5 µM EDTA PBS (ice-cold) containing counting beads. Samples were centrifuged (300 g, 5 min) and red blood cells were lysed three times with 1 mL ACK buffer (Gibco), washing with 1 mL PBS and centrifuging (300 g, 5 min) between each ACK lysis. Cells were blocked for 10 min using TruStain (also known as an Fc blocker or anti-mCD32 / 16). Cells were washed with FACS buffer and then stained with a mixture of antibodies containing markers of the following major white blood cell populations: monocytes (CD11b+, Ly6C+, Ly6G-), monocyte-derived dendritic cells (CD45+, CD11b+, CD11c+, MHCII+), neutrophils (CD11b+, Ly6G+), eosinophils (SSChi, CD11b+, Siglec F+), basophils (CD45lo, CD131+), NK cells (CD45+, NK1.1+), T cells (CD45+, CD3+, MHCII-), and B cells (CD45+, CD19+, MHCII+).

[0175] Peritoneal lavage fluid

[0176] Peritoneal lavage fluid was harvested using ice-cold 5 µM EDTA PBS containing counting beads. The sample was centrifuged and blocked with TruStain (as above). Next, the cells were washed and mast cells (CD45+, FcεRI+, and c-Kit+ (clone 2B8)) were stained.

[0177] marrow

[0178] A whole femur was harvested and kept in ice-cold 5 µM EDTA PBS. The bone was pulverized using a mortar and pestle with an EDTA-PBS solution containing counting beads. The total liquid was recovered and passed through a 70 µm filter chamber to remove bone and other tissue debris. The sample was centrifuged at (300 g, 5 min). The supernatant was removed, and erythrocytes were lysed (as above). Cells were then stained with the following antibody mixture: lineages (CD11b+, B220+, Ter119+, CD3+, Ly6G+, Ly6C+), cKIT, Sca-1 (Ly6A), CD34, IL-7Rα, CD16 / 32. Quantification was performed on lineage-Kit+ (LK) cells and lineage-Sca-1+Kit+ (LSK) cells, which may have been affected by different treatments. cKIT expression was assessed using antibody clone 2B8, which does not compete with clone ACK2. Lineage-Sca-1+Kit- cells were used as an internal control because these cells should not be directly affected by any anti-cKIT antibody treatment.

[0179] The scope of this invention is not limited to the specific embodiments described herein. In fact, many modifications to the invention (in addition to those described herein) will become apparent to those skilled in the art from the foregoing description and drawings. Such modifications are intended to fall within the scope of the appended claims. Furthermore, all aspects and embodiments of the invention described herein are considered broadly applicable and can be combined with any and all other consistent embodiments, including those derived where appropriate from other aspects of the invention (including individually).

[0180] This article cites multiple publications and patent applications, the disclosures of which are incorporated in their entirety through citation.

Claims

1. A method for depleting mast cells in a subject, the method comprising administering an effective amount of an anti-mast cell antibody to the subject, wherein the anti-mast cell antibody is administered to the subject via a slow absorption delivery route.

2. A method for treating mast cell disease in a subject in need, the method comprising administering a therapeutically effective amount of an anti-mast cell antibody to the subject, wherein the anti-mast cell antibody is administered to the subject via a slow absorption delivery route.

3. The method of claim 1 or claim 2, wherein the antimast cell antibody is administered subcutaneously or intradermally.

4. The method of claim 3, wherein the anti-mast cell antibody is administered subcutaneously.

5. The method of claim 4, wherein the anti-mast cell antibody is administered via a subcutaneous implant.

6. The method of claim 1 or claim 2, wherein the antimast cell antibody is not administered intravenously.

7. The method of any one of the preceding claims, wherein the anti-mast cell antibody specifically binds to a protein expressed on the surface of mast cells.

8. The method of any one of the preceding claims, wherein the anti-mast cell antibody is an anti-c-KIT antibody.

9. The method of any one of the preceding claims, wherein the anti-mast cell antibody comprises an Fc region.

10. The method of claim 9, wherein the Fc region has one or more Fc effector functions.

11. The method of claim 9, wherein the Fc region has one or more enhanced Fc effector functions relative to the wild-type Fc region.

12. The method of claim 10 or claim 11, wherein the one or more Fc effector functions are selected from: antibody-dependent cytotoxicity (ADCC); antibody-dependent phagocytosis (ADCP); and complement-dependent cytotoxicity (CDC).

13. The method of claim 12, wherein the Fc region has Fc effector function antibody-dependent cytotoxicity (ADCC).

14. The method of claim 12, wherein the Fc region has enhanced Fc effector function antibody-dependent cytotoxicity (ADCC) relative to the wild-type Fc region.

15. The method of any one of the preceding claims, wherein the anti-mast cell antibody is a human IgG antibody.

16. The method of claim 15, wherein the Fc region comprises amino acid substitution S239D / I332E.

17. The method of any one of claims 9 to 16, wherein the Fc region is non-fucosylated.

18. The method of any one of claims 9 to 17, wherein the Fc region is galactosylated.

19. The method of any one of claims 2 to 18, wherein the mast cell disease is mastocytosis.

20. The method of any one of the preceding claims, wherein the object has an abnormally high number of mast cells.

21. The method of any one of the preceding claims, wherein the object is a person.

22. The method of any one of the preceding claims, wherein the anti-mast cell antibody is formulated in a pharmaceutical composition.

23. The method of claim 22, wherein the pharmaceutical composition is formulated for slow absorption of the anti-mast cell antibody.

24. The method of claim 23, wherein the pharmaceutical composition is a modulated release formulation selected from slow-release, prolonged-release, long-acting-release, sustained-release, delayed-release, or controlled-release formulations.

25. The method of any one of the preceding claims, wherein the method reduces or prevents mast cell activation or mast cell degranulation.

26. The method of any one of the preceding claims, wherein the method reduces or prevents allergic or anaphylactic reactions.

27. The method of any one of the preceding claims, wherein the administration of the anti-mast cell antibody does not induce mast cell degranulation, allergic reaction, and / or anaphylactic reaction.

28. An anti-mast cell antibody used for treating mast cell disease by any one of claims 1 to 27.

29. Use of anti-mast cell antibodies in the preparation of medicaments for treating mast cell diseases, wherein the treatment is performed according to any one of claims 1 to 27.

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